The perfection of surface fluorometry for measuring local metabolic states and for discerning the profiles of hypoxic regions has won considerably greater acceptance in perfused models, where the precision is high and the possibilities of artifacts few, and in situ organs, where care is required lest blood flow and blood volume changes in the area of the recording give spurious readings. In the latter case, satisfactory readout of reflectance signals at appropriate wavelengths, together with algorithms for applying a correction factor, lead to a remarkable clarity of recording, in spite of large hemodynamic changes. In particular, the instruments have been designed to record at a single point, or, more accurately, in an area from 10 microns to several millimeters in diameter. While this has been eminently satisfactory for giving a report on the general state of the area under observation, particularly in response to ischemia and hypoxia, the combined technique of freeze-trapping and low temperature scanning has suggested a considerable heterogeneity of the redox states of the brain cortex, heart, and liver, indicating microheterogeneity of the metabolic activity and/or of oxygen delivery to various parts of the organ. While the low temperature scanning procedure gives a " stop motion" picture of the phenomenon with considerable accuracy and resolution and the possibility of three-dimensional reconstruction, there is a need to be able to carry out on-line studies of distributions of redox states in a given area involved, for example, in a localized hypoxia in heart or brain. The goals may be either to identify an appropriate heterogeneous state for freeze-trapping and further examination of "maximal deviation" metabolic state, or to read off as a function of time the effects on the size of a hypoxic region in a normoxic tissue of such parameters as oxygen tension substrates, inhibitors or pharmacologic reagents.
Two approaches to a two-dimensional scan of tissue fluorescence have already been tried: first, the television approach with varying degrees of sophistication in the camera, i.e., image amplifiers, image converters, etc. Secondly, image amplifiers can be focussed on charge-coupled devices with considerable improvement in dynamic range, stability, etc. Preliminary tests of the latter configuration have been made. The performance of these techniques is not enhanced by the use of laser excitation of fluorescence, simply because the average power of a laser may not be very high, as compared with that of 1 KW Hg or Xenon (used with appropriate filters), when broadcase illumination over the sample is employed.
The flying spot scanner has previously been used with cathode ray tubes as light sources, and becomes advantageous for fluorescence excitation when a laser is used as the light source. For the past several years, cheap and efficient laser light sources have been available at wavelengths appropriate to oxidized flavoprotein and reduced pyridine nucleotide, and comparisons of the various lasers with mercury arcs, etc. have been made, particularly the He-Cd laser.